A Method of transferring a sample out of a first state into a second state using an optical signal is known from German patent application DE 101 54 699 A1. For investigating a sample by fluorescence microscopy, the fluorescence marker molecules in the sample are first brought into an excited energy state using an exciting optical signal. With regard to this optical excitation the usual limit for spatial resolution in optical methods of λ/2n applies, wherein λ is the wavelengths of the light used and n is the diffraction index of the sample. To improve the spatial resolution beyond this limit, the optically excited state is depleted with a de-exciting optical signal everywhere in the sample, except at desired measuring points in which the de-exciting optical signal intensity distribution features a local zero. For example, the fluorescence marker molecules in the sample are quenched by stimulated emission using the de-exciting optical signal. This is the case outside the measuring point where the de-excitation optical signal is not zero or not close to zero. The dimensions of the resulting fluorescent measuring point, i.e. the spatial resolution of the remaining fluorescence, can be reduced much below the common optical resolution limit, in that the de-exciting optical signal is applied to the sample outside the desired measuring point at such an intensity that a saturation is achieved in depleting the fluorescent energy state by stimulated emission. Thus, the fluorescent marker molecules in the sample are in the fluorescent state only in a very narrowly limited area around the zero point of the intensity distribution of the de-exciting optical signal. According to the publication Hell, Nature Biotechn., 21, 1347–1355, the size of the fluorescent measuring point Δx and thus the spatial resolution follow Δx≈λ/(2n√{square root over (I/Is)}), wherein λ is the wavelengths, n is the diffraction index, I is the applied intensity and Is is the saturation intensity. The saturation intensity is the characteristic intensity at which the sample is de-excited by 50% because of the influence of the de-exciting optical signal from a probabilistic point of view. The saturation intensity Is is inversely proportional to the cross-section σ of the de-exciting optical signal according to Is=1/(στ). Here, τ is the average life time for which the sample remains in the excited state, before it spontaneously decays into the de-excited state. As a result, the following applies to the resolution Δx≈λ/(2n√{square root over (Iστ)}). Thus for achieving a high resolution it is advantageous to work with an intensity of the de-exciting optical signal that is as strong as possible, with a cross-section σ that is as large as possible and with a fluorescent state featuring a life time τ that is long enough.
Maximizing the intensity of the de-exciting optical signal, which is required for achieving the saturation, there is the danger, that the sample is chemically modified. Such a chemical modification, as a rule, is caused by a formation of radicals due to the strong light intensities, which enable various chemical reactions. Particularly, if oxygen and other reactive species are in the sample, the de-exciting optical signal causes undesired chemical reactions that are known as “photobleaching ” when they affect the fluorescence dye in the sample. The intensity of the de-exciting optical signal necessary for saturation could be reduced, if the cross-section σ could be increased.
However, it is often necessary to work at a wavelength of the de-exciting optical signal at which σ is comparatively low. The reason is that at another, often shorter wave length at which the cross-section would be higher, other undesired processes would be initiated besides the desired de-excitation. These other processes would interfere with the desired de-excitation of the marker molecules in the sample, like for example, they would excite the sample into the excited fluorescent state again so that the overall efficiency of the desired de-excitation would be decreased.
The lifetime τ of a certain excited fluorescent state is generally fixed.
From J. Jasny et al.: “Fluorescence microscopy in superfluid helium: single molecule imaging” Re. Sci. Instrum 67 (4), April 1996, pages 1425–1430 it is known that low temperatures (of about 2 K) in detection and spectroscopy of single molecules have the result that the full width at half maximum of the absorption line from a ground state to the lowest excited singlet state of the observed molecule becomes so narrow that it reaches the limits set by its natural lifetime. As a result, environmental influences on the molecule result in a shift of the absorption line of much more than its full width at half maximum. Observing the line thus allows for spectroscopic monitoring of various environmental influences on the molecule.
A need still exists for a method of fluorescence-microscopically investigating a biological sample at high spatial resolution without damaging the sample with the high intensities of the de-exciting optical signal.